DNA barcoding of Telmatherinidae family in Lake Towuti, South , 1Jayadi Jayadi, 1Ilmiah Ilmiah, 1Siti Hadijah, 1Muhammad Kasnir, 2Dewi I. Roslim

1 Department of Aquaculture, Faculty of Fishery and Marine Science, Indonesia Muslim University, Makassar, Indonesia; 2 Department of Biology, Faculty of Mathematics and Nature Sciences, Universitas Riau, Pekanbaru, Indonesia. Corresponding author: J. Jayadi, [email protected]; [email protected]

Abstract. Telamtherinidae is a family of endemic in South Sulawesi. The study is aimed at identifying the molecular gene of the endemic fish of the Telmatherinidae family in Towuti Lake, South Sulawesi, to identify and analyze genetic diversity, genetic marking, genetic distances, genetic characterization, and dendrogram of the fish. DNA barcoding in this research used the Cytochrome c Oxidase I (COI) gene. Amplification of mitochondrial COI gene regions was conducted by using COI Fish F2 and COI Fish R 2 primer. Data analysis, total isolation of DNA, Polymerase Chain Reaction (PCR), Electrophoresis, gel purification, and sequencing Basic Local Alignment Search Tool (BLAST) were performed. The results showed that DNA sequence was 681 bp. Meanwhile, analysis of dendrogram suggested that the fish of Telmatherinidae family in Towuti Lake are similar to the fish in the , and Tominanga genera. The genus Telmatherina including Telmatherina celebensis, Telmatherina bonti, and Telmatherina opudi showed 85% significant shared similarity with Atherina sp. Only 84% similarity of genus Telmatherina was found in Pristigenys alta, Parexocoetus brachypterus, Cypselurus hiraii, and Hypoatherina tsurugae. Meanwhile, genus Paratherina: Paratherina striata and Paratherina wolterecki have 83-84% similarity with Scorpis lineolata, Hyporhamphus affinis, Cypselurus hiraii, Parexocoetus brachypterus, and Pristigenys alta. In addition, genus Tominanga: Tominanga sanguicauda has 84% similarity with Hypoatherina tsurugae, Cypselurus hiraii, Prognichthys sealei, Hyporhamphus affinis, and Pristigenys alta. Key Words: genetic diversity, Telmatherinidae, COI gene, dendrogram, Lake Towuti.

Introduction. Lake Towuti is located in Nuha sub-district, East Luwu regency South Sulawesi, Indonesia, and is interconnected with Lake Matano and Lake Mahalona. These three lakes are included in an ancient Lake Complex (Wijaya et al 2009; Samuel et al 2009; Nasution et al 2010; Umar et al 2012). Lake Towuti has been an important habitat for several endemic freshwater fish (Kottelat et al 1993; Wirjoatmodjo et al 2003; Samuel et al 2009; Parenti 2011; Parenti & Ebach 2013) such as the fish families from Telmatherinidae, Gobidae, Adrianichthyidae and Hemiramphidae (Herder et al 2006; Nasution et al 2007; Stelbrink et al 2014; Hutama et al 2016). They are economically important for many local communities around Lake Towuti (Wijaya et al 2009). Potencial productivity of fish in Lake Towuti is equal to ±195 ton year-1 (Wijaya et al 2009). Some endemic fish species are utilized for consumption and ornamental purposes. Consequently, the endemic fish population continues to decline yearly, and conservation measures have not been carried out properly (Mamangkey et al 2007; Wijaya et al 2009; Samuel et al 2009; Nasution et al 2010; Umar et al 2012). The uncontrolled exploitation of endemic fish can lead endemic fish into extinction, even in their natural habitat (Jayadi et al 2015, 2016; Nasution et al 2015). Another threat that causes a decline of endemic fish populations in Lake Towuti is water pollution from domestic waste disposal. Pollution detrimentally affects the environment as well as endemic fish. Moreover, land clearing for settlement and agriculture around the lake leads to erosion and sedimentation. This is exacerbated by introducing invasive fish species into Lake Towuti (Prianto et al 2014).

AACL Bioflux, 2019, Volume 12, Issue 4. 1208 http://www.bioflux.com.ro/aacl Several endemic fish species included in the category of vulnerable species in Lake Towuti include Termatherinidae family of genera Telmatherina, Paratherina, and Tominanga (Kottelat et al 1993; IUCN 2003; Suwelo 2005; Herder et al 2006; Nasution et al 2007; Stelbrink et al 2014; Hutama et al 2016). The Telmatherinidae family was referred to as sailfin silversides fish (Kottelat 1991; Stelbrink et al 2014; Hutama et al 2016). Therefore, it is necessary to conduct sustainable management, such as genetic conservation of species and habitat. Genetic conservation is one of sustainable management measures that can be applied by analyzing the genetic diversity of particular species (Mamangkey et al 2007; Hadijah et al 2014; Jayadi et al 2015). Genetic diversity analysis provides the short and long term information related to a particular species’ population (Ferguson et al 1995). For instances, genetic diversity can be used in considering biological resource management (Yusron 2005; Jayadi et al 2015; Nugroho et al 2017). Furthermore, it can be used for improving fish stock (Islam et al 2011), domestication, and aquaculture (Lante et al 2011; Iskandariah et al 2015; Lorenzen et al 2012; Jayadi et al 2016). To analyze the genetic diversity, barcoding DNA using COI can be applied (Ward et al 2005; Muchlisin et al 2013; Jusmaldi et al 2014; Hubert et al 2015; Nuryanto et al 2017; Pramono et al 2017; Abbas et al 2017). The purpose of this study is to determine genetic diversity using molecular identification such as the COI DNA gene in the endemic species of the Telmatherinidae family in Lake Towuti as well as genetic marking, genetic characters, and dendrogram.

Material and Method

Fish sampling. Fish sampling for Telmatherinidae fish family was conducted in Lake Towuti, South Sulawesi, Indonesia from January to June, 2018, using fishing net size of 1 mm. Morphological identification was carried out according to Herder et al (2006), Kottelat (1991), Kottelat et al (1993), and Said & Hidayat (2015). In addition, fish muscle (from 15 fish) in the tail was taken (1 cm x 1 cm) and then placed into a 96% ethanol solution and stored in the freezer.

Total DNA isolation. D NEasy Blood and tissue kit (cat. No. 69504 and 69506 Qiagen) was applied for total DNA. The total DNA isolation process was conducted based on the existing protocol. The electrophoresis technique was performed to measure the quality and the quantity of the total DNA.

Polymerase Chain Reaction (PCR). Total DNA was amplified by PCR technique using a pair of universal primers for COI genes in fish, namely Fish F2: 5'-TCG ACT AAT CAT AAA GAT ATC GGC AC-3 'and FishR2: 5' TCA ACT GGG TGA CCG AAG AAT CAG AA -3' (Ward et al 2005). The PCR components include 1X of Supreme NZY Taq, 2X Green Master Mix,

2.4 µM of forward primary, 2.4 µM reverse primer, 1 µL of total DNA, and dH2O until 50 µL of PCR volume. The PCR program includes pre-PCR at 94oC for 5 minutes; PCR was 35 cycles consisting of denaturation at 94oC for 45 seconds, annealing at 50oC for 45 seconds, elongation at 72oC for 1 minute, and post-PCR at 72oC for 10 minutes. PCR success was detected by electrophoresis techniques.

Electrophoresis. Electrophoresis was performed to detect the success of DNA and PCR isolation. Total DNA and PCR products were moved to a 1% agarose gel in a 1X TBE buffer at 50 voltage for 45 minutes. DNA tape was colored using 5 µg mL-1 of ethidium bromide, visualized on a UV transilluminator lamp, and then photographed using a UV filtered digital camera.

DNA sequencing. DNA sequencing was performed at PT Genetika Science in Jakarta as a channeling agent. Gene purification and sequencing were carried out at 1st Base in Malaysia. The PCR product was 40 µL and each primer was 30 µL.

AACL Bioflux, 2019, Volume 12, Issue 4. 1209 http://www.bioflux.com.ro/aacl Data analysis. DNA sequence data was used forward and reverse primers which were put together or aligned using BioEdit7 software. The BLAST (Basic Local Alignment Search Tool) analysis at http://www.ncbi.nih.nlm.gov/BLAST (Madden 2013) was carried out on DNA sequences from each fish sample to determine its similarity to DNA COI sequences in the GenBank database. Accessions were of high similarity, and sequences were downloaded to make phylogenetic trees using the MEGA (molecular evolutionary genetics analysis) version 6.06 (build #: 6140226) (Tamura et al 2013) based on the Kimura 2-parameter model and UPGMA (Unweighted Pair Group Method with Arithmetic mean) with 1000 bootstrap.

Results and Discussion. Amplification of fish samples from the Telmatherinidae family using primary Fish_F2/Fish_R2 produced 750 bp of DNA tape (Figure 1). The DNA band that has been produced indicates that the sequencing process was done properly. The DNA sequence obtained from 20 samples was 681 bp and registered in Gen Bank (Table 1).

Figure 1. Profiles of DNA bands that use Fish_F2 / Fish_R2 primers. L = 1 kb DNA Ladder (Thermo Scientific).

Table 1 Registration number of DNA sequence results of 20 fish samples from the family of Telmatherinidae in Lake Towuti

No Fish spesies Sample label Registration number 1 Telmatherina celebensis A01 MH568798 2 Telmatherina celebensis A02 MH568799 3 Paratherina striata B01 MH568800 4 Paratherina striata B02 MH568801 5 Tominanga sanguicauda CO1 MH568802 6 Tominanga sanguicauda CO2 MH568803 7 Tominanga sanguicauda CO3 MH568804 8 Paratherina wolterecki D10 MH568805 9 Paratherina wolterecki D11 MH568806 10 Paratherina wolterecki D12 MH568807 11 Telmatherina bonti E13 MH568808 12 Telmatherina bonti E14 MH568809 13 Paratherina cyanea F15 MH568810 14 Paratherina cyanea F16 MH568811 15 Paratherina cyanea F17 MH568812 16 Telmatherina opudi G18 MH568813

DNA barcodes of endemic fish in the Telmatherinidae family in Lake Towuti using primary COI Fish_F2 / Fish_R2 found long DNA band fragments after mt-DNA amplification to around 681 bp. The COI primer has been applied to identify the genus Thunnus and

AACL Bioflux, 2019, Volume 12, Issue 4. 1210 http://www.bioflux.com.ro/aacl genus Squalus with a fragment length of 655 bp (Ward et al 2005), the genus Kryptopterus around 707 bp (Jusmaldi et al 2014), and the genus Mystus at 697 bp (Pramono et al 2017). The length of fragments in endemic fish in South Sulawesi such as Glossogobius matanensis was 500 bp (Mamangkey et al 2007), Glossogobius aureus was 600 bp (Hadijah et al 2014), Telmatherina ladigesi was 600 bp (Jayadi et al 20015), Pterygoplichthys sp. was 650 bp (Rosnaeni et al 2017), Family Sparinidae was 650 bp (Abbas et al 2017), and Harpadon nehereus, Harpadon microchir and Harpadon squamosus were 618 bp (Nugroho et al 2017). DNA barcoding is used to assign a biological specimen to a species (Ardura et al 2010; Fahmi et al 2016; Nuryanto et al 2017; Pramono et al 2017). Furthermore, DNA barcoding contributes to science and promotes more sustainable practices in and the development of new molecular tools for species identification (Hubert et al 2015). The results of the Telmatherinidae family dendrogram analysis obtained three genera, namely: Paratherina, Telmatherina, Tominanga (Figure 2).

Figure 2. Dendrogram in the Telmatherinidae family in Lake Towuti.

Phylogenetic results show that three genera from the Telmatherinidae family (Paratherina, Telmatherina and Tominanga) were found in Lake Towuti. Species of the genus Paratherina are Paratherina striata, Paratherina wolterecki, Paratherina cyanea, and the genus Telmatherina is represented by Telmatherina celebensis, Telmatherina bonti, Telmatherina opudi, and the genus Tominanga is represented by Tominanga sanguicauda (Figure 2). All types of fish have been registered in Gen Bank (Table 1). Those fish population are known as native and endemic in Lake Towuti (Kottelat 1991; Parenti 2011; Stelbrink et al 2014; Hutama et al 2016). The population of fish species from the Telmatherinidae family in Lake Towuti has decreased (Kottelat et al 1993; Suwelo 2005; Herder et al 2006; Nasution et al 2007; Stelbrink et al 2014; Hutama et al 2016) and have become vulnerable species (IUCN 2003). Consequently, inbreeding in the population leads to lower genetic variation of Telmatherinidae family. Low value of genetic diversity shows that a narrow level of migration can provide opportunities for limited gene exchange with other populations (Sugama et al 1996). Genetic variation of fish populations in nature can reduce due to damaged habitat, limited migration, isolated from other populations, depression inflation and decreased reproductive ability (Jayadi et al 2015). Changes in genes can occur due to individual gene migration in a population (Ezilrani & Christopher 2015). In addition, genetic variation is important for evaluating fish resources in the wild. Genetic variations have a direct or indirect potential impact on populations, communities, and ecosystems (Hughes et al 2008). As said by Ezilrani & Christopher (2015), the genetic structure of a fish species is an illustration of the long changes due to biological and environmental factors. The results of the BLAST-GEN analysis for endemic fish of Telmatherinidae family in the Gen Fish Bank are presented in Table 2.

AACL Bioflux, 2019, Volume 12, Issue 4. 1211 http://www.bioflux.com.ro/aacl Table 2 Analysis of BLAST-GEN in the Telmatherinidae family

Max Total Query E Fish species Description Indent Accession score score cover value Telmatherina Atherinidae sp. 673 673 95% 0.0 85% MF409492.1 celebensis Scorpis lineolata 685 685 99% 0.0 84% AP011063.1 Pristigenys alta 673 673 99% 0.0 84% MG856600.1 Hypoatherina tsurugae 673 673 99% 0.0 84% AP004420.1 Cypselurus hiraii 667 667 99% 0.0 84% AB182653.1 Paratherina Scorpis lineolata 673 673 99% 0.0 84% AP011063.1 striata Hyporhamphus affinis 656 656 98% 0.0 84% KJ013045.1 Cypselurus hiraii 656 656 99% 0.0 83% AB182653.1 Parexocoetus brachypterus 650 650 99% 0.0 83% KY067947.1 Pristigenys alta 644 644 99% 0.0 83% MG856600.1 Tominanga Hypoatherina tsurugae 685 685 99% 0.0 84% AP004420.1 sanguicauda Cypselurus hiraii 679 679 99% 0.0 84% AB182653.1 Prognichthys sealei 673 673 99% 0.0 84% KY067951.1 Hyporhamphus affinis 667 667 98% 0.0 84% KJ013045.1 Pristigenys alta 662 662 99% 0.0 84% MG856600.1 Paratherina Pristigenys alta 685 685 99% 0.0 84% MG856600.1 wolterecki Cypselurus hiraii 685 685 99% 0.0 84% AB182653.1 Hypoatherina tsurugae 679 679 99% 0.0 84% AP004420.1 Cheilopogon doederleinii 673 673 99% 0.0 84% AP017897.1 Cheilopogon arcticeps 673 673 99% 0.0 84% KU360728.1 Telmatherina Scorpis lineolata 673 673 95% 0.0 85% MF409492.1 bonti Hyporhamphus affinis 679 679 99% 0.0 84% MG856600.1 Cypselurus hiraii 679 679 99% 0.0 84% KY067947.1 Parexocoetus brachypterus 673 673 99% 0.0 84% AB182653.1 Pristigenys alta 673 673 99% 0.0 84% AP004420.1 Paratherina Pristigenys alta 685 685 99% 0.0 84% MG856600.1 cyanea Cypselurus hiraii 685 685 99% 0.0 84% AB182653.1 Hypoatherina tsurugae 679 679 99% 0.0 84% AP004420.1 Cheilopogon doederleinii 673 673 99% 0.0 84% AP017897.1 Cheilopogon arcticeps 673 673 99% 0.0 84% KU360728.1 Telmatherina Atherinidae sp. 673 673 95% 0.0 85% MF409492.1 opudi Pristigenys alta 679 679 99% 0.0 84% MG856600.1 Parexocoetus brachypterus 679 679 99% 0.0 84% KY067947.1 Cypselurus hiraii 673 673 99% 0.0 84% AB182653.1 Hypoatherina tsurugae 673 673 99% 0.0 84% AP004420.1

Table 2 illustrates that the genus Telmatherina including Telmatherina celebensis, Telmatherina bonti and Telmatherina oputi showed 85% shared significant similarity to Atherinidae sp. Only 84% similarity of genus Telmatherina was found in Pristigenys alta, Parexocoetus brachypterus, Cypselurus hiraii, and Hypoatherina tsurugae. Meanwhile, genus Paratherina: Paratherina striata, Paratherina wolterecki have similarities between 83 and 84% with Scorpis lineolata, Hyporhamphus affinis, Cypselurus hiraii, Parexocoetus brachypterus, and Pristigeny alta. In addition, genus Tominanga: Tominanga sangicauda has 84% similarity to Hypoatherina tsurugae, Cypselurus hiraii, Prognichthys sealei, Hyporhamphus affinis, and Pristigenys alta.

Conclusions. The use of the cytochrome c oxidase I (COI) DNA has been successful in identifying the family of endemic fish from Telmatherinidae family in Lake Towuti, South Sulawesi Indonesia. The research finding shows that the genus Paratherina is represented by the species Parathrina striata, Paratherina wolterecki, Paratherina cyanea, the genus Telmatherina - Telmatherian celebensis, Telmatherina bonti, Telmatherina opudi, and the genus Tominanga - Tominanga sanguicauda.

Acknowledgements. The authors would like to thank the Directorate Research and Community Service, Ministry of Research, Technology and Higher Education for funding this research through Scheme on Basic Research of Higher Education Research Grants in 2018. We also thank to Institutions of Research and Community Service, Muslim Indonesia University and Department of Biology Faculty of Mathematics and Nature Science of Riau University for analysis of genetics.

AACL Bioflux, 2019, Volume 12, Issue 4. 1212 http://www.bioflux.com.ro/aacl References

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Received: 01 April 2019. Accepted: 23 June 2019. Published online: 21 August 2019. Authors: Jayadi Jayadi, Department of Aquaculture, Faculty of Fishery and Marine Scince, Universitas Muslim Indonesia, Jl. Urip Sumoharjo Km 05, Makassar 9023, Indonesia, e-mail: [email protected]; [email protected] Ilmiah Ilmiah, Department of Aquaculture, Faculty of Fishery and Marine Scince, Universitas Muslim Indonesia, Jl. Urip Sumoharjo Km 05, Makassar 9023, Indonesia, e-mail: [email protected] Siti Hadijah, Department of Aquaculture, Faculty of Fishery and Marine Scince, Universitas Muslim Indonesia, Jl. Urip Sumoharjo Km 05, Makassar 9023, Indonesia, e-mail: [email protected] Muhammad Kasnir, Department of Aquaculture, Faculty of Fishery and Marine Scince, Universitas Muslim Indonesia, Jl. Urip Sumoharjo Km 05, Makassar 9023, Indonesia, e-mail: [email protected] Dewi Indriyani Roslim, Department of Biology, Faculty of Mathematics and Nature Sciences, Universitas Riau, Pekanbaru, Indonesia, e-mail: [email protected] This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited. How to cite this article: Jayadi J., Ilmiah I., Hadijah S., Kasnir M., Roslim D. I., 2019 DNA barcoding of Telmatherinidae family in Lake Towuti, South Sulawesi, Indonesia. AACL Bioflux 12(4):1208-1215.

AACL Bioflux, 2019, Volume 12, Issue 4. 1215 http://www.bioflux.com.ro/aacl